Male infertility affects 5-10% of the population. A major factor associated with male infertility is Y chromosome deletions, yet our understanding of the requirement for specific genes on the Y chromosome and of their roles in sperm production/function is still poor. Y chromosome genes may provide essential spermatogenic functions or just potentiate the spermatogenic process. Our long-term goal is to define the function of Y chromosome encoded genes in mice in a context of assisted reproduction technologies (ART) as a way to model human Y- linked infertility cases. We have established that only two Y chromosome genes, testis determinant gene Sry and spermatogonial proliferation factor Eif2s3y are required for production of male gametes capable of participating in assisted fertilization. In Preliminary Data we show that males with a Y complement limited to Sry and Eif2s3y have spermatogenesis arrest and do not produce mature sperm. The precursor haploid germ cells (spermatids) are rare and often abnormal. Nevertheless, with round spermatid injection (ROSI) we succeeded in producing viable, healthy, and fertile progeny. This offers a promise to men with extensive Y gene loss and resulting azoospermia. Here, our specific goal is to define whether ART can be achieved even without this minimum Y gene contribution. We will test the hypothesis that the Y chromosome complement can be eliminated entirely while retaining production of functional male gametes. In Aim 1 we wil test if transgenic activation of Sox9, a downstream effector of Sry, will effectively replace Sry function and whether Sry-to-Sox9 replacement affects spermatogenesis and fertility, testing directly for the as yet unknown function of these genes in mature gonads. We will also establish if Eif2s3x, an X encoded homologue of Eif2s3y, can replace Eif2s3y function in spermatogonial proliferation. We will generate and characterize mice transgenic for Eif2s3x, and assess whether overexpression of Eif2s3x can rescue spermatogonial proliferation arrest in XOSry mice. In Aim 2, we will produce males without any Y genes but with overexpression of Eif2s3x and with transgenic activation of Sox9, as well as males with one Y gene retained and the other replaced. We will investigate how the presence of these genes affects spermatogenesis progression. We will also test if spermatogenesis in these males enables development of germ cells functional in ART, and whether such produced offspring are normal. In Aim 3, we will attempt to rescue spermatogonial arrest in testes of mature males with in vivo Eif2s3y gene transfer using novel 'active transgenesis' approach and ultrasound mediated gene delivery. Our studies will advance the understanding of (1) the roles that key players of sex determination (Sry and Sox9) play in mature gonads; (2) the roles of sex chromosome genes (Eif2s3y and Eif2s3x) in the initiation of spermatogenesis; and (3) the compatibility of extensive Y gene loss with successful ART. Our results should translate to enhance treatment of human infertility associated with Y chromosome deletions. ! PUBLIC HEALTH RELEVANCE: The project will test, in a mouse model, whether males lacking the entire Y chromosome can generate functional gametes and father healthy offspring with assisted reproduction, in spite of their obvious infertility in normal fertilization. This wil be of importance for infertile men with extensive Y chromosome deletions, which are a target group of human ART. The project will also advance the understanding of the roles of Y chromosome encoded genes in spermatogenesis.